Basic and Clinical July, August 2020, Volume 11, Number 4

News and Reports: Identification of the rs797045105 in the SERAC1 by Whole-exome Sequencing in a Patient Suspicious of MEGDEL Syndrome

Mina Zamani1,2 , Tahereh Seifi1,2, Jawaher Zeighami1, Neda Mazaheri1,2, Emad Jahangirnezhad1, Minoo Gholamzadeh1, Alireza Sed- aghat1,3* , Gholamreza Shariati1,4* , Hamid Galehdari1,2

1. Narges Medical Genetics and Prenatal Diagnosis Laboratory, Ahvaz, Iran. 2. Department of Genetics, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran. 3. Diabetes Research Center, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. 4. Department of Genetics, School of Medicine, Ahvaz Jundishapur University of medical Sciences, Ahvaz, Iran.

Use your device to scan and read the article online Citation: Zamani, M., Seifi, T., Zeighami, J., Mazaheri, J., Mazaheri, N., & Jahangirnezhad, F., et al. (2020). Identification of the rs797045105 in the SERAC1 Gene by Whole-exome Sequencing in a Patient Suspicious of MEGDEL Syndrome. Basic and Clinical Neuroscience, 11(4), 549-556. http://dx.doi.org/10.32598/bcn.9.10.455

: http://dx.doi.org/10.32598/bcn.9.10.455

A B S T R A C T

Introduction: Whole Exome Sequencing (WES) has been increasingly utilized in genetic determinants of various inherited diseases. Article info: Received: 13 Oct 2017 Methods: We applied WES for a patient presenting 3-Methylglutaconic Aciduria (MEG), First Revision: 10 Nov 2017 Deafness (D), (E), and Leigh-like (L) syndrome. Then Sanger sequencing was used for the detected variant validation. Accepted: 13 May 2019 Available Online: 01 Jul 2020 Results: We found an insertion, rs797045105 (chr6, 158571484, C>CCATG), in the SERAC1 gene with homozygous genotype in the patient and heterozygous genotype in her unaffected parents. Notably, bioinformatics analysis using mutation taster (prob>0.99) and DDIGin (prob=86.51) predicted this mutation as disease-causing. Also, the variant was not present in our database, including 700 exome files. Keywords: Conclusion: These findings emphasize the pathogenicity of rs797045105 for MEGDEL syndrome. On the other hand, our data shed light on the significance of WES application as a Whole-exome sequencing, genetic test to identify and characterize the comprehensive spectrum of genetic variation and rs797045105, SERAC1, classification for patients with neuro- metabolic disorders. MEGDEL

* Corresponding Author: Hamid Galehdari, PhD. Gholamreza Shariati, PhD. Address: Department of Genetics, Faculty of Science, Shahid Chamran Address: Department of Genetics, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz, Iran. University of Ahvaz, Ahvaz, Iran. Tel: +98 (916) 6149028 Tel: +98 (912) 1115529 E-mail: [email protected] E-mail: [email protected]

549 Basic and Clinical July, August 2020, Volume 11, Number 4

Highlights

● WES is an applicable tool to evaluate genetically metabolic diseases.

● rs797045105 highly potentially causes MEGDLE syndrome.

● rs797045105 makes a frameshift change in SERAC1 gene.

Plain Language Summary

The genetic architecture of metabolic traits and diseases is complex. Many have role in the pathogenicity of these diseases. Advanced technologies including Whole Exome Sequencing (WES) which can assess many genes si- multaneously could be an effective option to find out the mutations causing heterogeneous metabolic disorders. In this study we applied WES for a patient presenting MEGDLE syndrome: a neuro-metabolic disease. After data analysis we found rs797045105 as a disease causing genetic variant for the patient. This pathogenic variation should be considered in genetic profiling of patients with MEGDLE syndrome especially from Southwest of Iran.

1. Introduction In recent years, Next-Generation Sequencing (NGS) has improved our knowledge about the genetic and mo- -methylglutaconic aciduria (3-MGA-uria) is a lecular bases of this heterogeneous group of disorders. heterogeneous group of metabolic syndromes featured by elevated urinary excretion of Here, we used Whole-Exome Sequencing (WES) for 3-methylglutaconic and 3-methyl glutaric ac- genetic diagnosis of an Iranian patient with 3-MGA-uria, 3 ids (Wortmann et al., 2012). mental retardation, cerebellar atrophy, deafness, impairment, and spasticity symptoms, which are The MEGDEL syndrome (3-MGA-uria type IV) has been compatible with MEGDEL syndrome. introduced by clinical presentations, including neurological deterioration, involvement, senso- 2. Materials and Methods ry-neural hearing loss, encephalopathy, and (Phe- notype MIM number: 614739). MEGDEL syndrome is a 2.1. Patient/ case report recessive disorder of dystonia and deafness with Leigh-like syndrome (Wortmann et al., 2006). A patient with MEGDEL and his parents were recruit- ed from the southwest of Iran. MEGDEL syndrome is caused by mutations in serine active site containing 1 (SERAC1). Studies by Wortmann 2.2. Peripheral blood samples et al. on fibroblasts derived from patients showed that SE- RAC1 is localized at the interface between mitochondria For blood sampling, 10 ml of peripheral blood of the and . They showed that mutations in patient and his parents were collected in EDTA tubes. SERAC1 can abnormally increase the ratio of phosphati- 2.3. DNA extraction dylglycerol-34:1 to phosphatidylglycerol-36:1. Therefore, it is speculated that this leads to lower bis (mono-acyl-glyc- Genomic DNA was extracted using the standard salt- erol) phosphate levels and the accumulation of cholesterol ing-out protocol. The quality and quantity of the extract- in the perinuclear region. Finally, it is hypothesized that SE- ed DNA samples were checked by the gel electrophore- RAC1 can catalyze the first enzymatic reaction in the bis- sis and NanoDrop. phosphate(mono-acyl-glycerol) biosynthetic pathway and has a role in the phosphatidylglycerol remodeling pathways 2.4. Exome sequencing as it is involved in the transacylation-acylation reaction to produce phosphatidylglycerol-36:1. They concluded that The sample was subjected to WES using the Illumina- SERAC1 encoded participates in both mitochondri- HiSeq 2000 Genome Analyzer Platform (CNAG) by the al function and intracellular cholesterol trafficking (Wort-� Macrogen Company. mann et al., 2012).

550 Zamani, M., et al. (2020). rs797045105 is the MEGDEL-causing Variant. BCN, 11(4), 549-556. Basic and Clinical July, August 2020, Volume 11, Number 4

Table 1. Sequences of the used primers

Name Sequence

SERAC1-F TGTCCTTGAACAAAAAGTCAGTATGT

SERAC1-R CCTCAATTAATTCTTCAGGAACTTGG

SERAC1-RTprimer-F AAGGCTGTGACATTAGATACTC

SERAC1-RTprimer-R1 AGCAAATGGATTCCGCAGTATC

SERAC1-RTprimer-R2 TGACTTGCTCTTCCGTAGG

2.4. RNA extraction, Reverse transcription 2.6. DNA Sanger-sequencing

Total RNA was extracted using TRIzol® reagent (Life Ampliqons were directly sequenced by the ABI Technologies). Also, the complementary DNA was syn- PRISM 3700 Genetic Analyzer (Applied Biosystems). thesized by the PrimeScript™ RT reagent kit (Takara The sequences were analyzed with Chromas LITE 2.1.1 Bio Inc, Shiga, Japan). and then compared with the reference genomic sequence using BLASTN. Also, the presence of the detected mu- 2.5. Polymerase Chain Reaction (PCR) tation was confirmed by the parent DNA analysis and bi-directional sequencing. The targeted region of the SERAC1 gene on DNA and cDNA was amplified by PCR using primers designed 2.7. In-silico analysis by Oligo7 software (Table 1). The PCR was conducted using the Master Mix (Ampliqon, Denmark). The PCR Various tools are available for the prediction of patho- thermal program was as follows: 95°C for 5 min, 35 cy- genicity of variations in coding and non-coding regions. cles of 95°C for 30 s, 60 °C for 30 s, and 72°C for 30 s, In the present study, we used some of these tools for the and 72°C for 5 min. evaluation of disease-causing potentiality of the present insertion variation. The prediction of variant pathogenic- ity was done using Mutation taster and DDIGin.

Figure 1. Family pedigree

Zamani, M., et al. (2020). rs797045105 is the MEGDEL-causing Variant. BCN, 11(4), 549-556. 551 Basic and Clinical July, August 2020, Volume 11, Number 4

3. Result The metabolic test report for the proband was as fol- lows: The organic acids in the patient’s urine showed a 3.1. Clinical reports for the patient moderate excretion of 3-methyglutaric acid (16 mmol/ mol creatinine, normally not detected) and 3-methylglu- The enrolled family belonged to the Lor ethnicity of taconic acid (79 mmol/mol creatinine, normal<19). Iran. The proband in the family was a boy (Figure 1, II- 2) with 8 years of age. Parents (Figure 1, II-1, and II-2) The following magnetic resonance imaging results of were the first cousins. The first son of the family had the the brain for the proband were obtained: increased sig- same symptoms as proband. The proband represented nal intensity in the deep white matter of the brain in T2- developmental delay and regression, sensory-neural weighted images is more probably representative of the hearing loss, myoclonic epilepsy, 3-methylglutaconic immaturity of the neural migration; however, the basal aciduria, mental retardation, respiratory problems, jaun- ganglia with increased signal in T1-weighted images dice, dysphagia, and lesion in basal ganglia. possibly indicate the metabolic disease of the brain.

Table 2. Location and predicted pathogenicity of the variation. Data were extracted by the Mutation taster and DDIG-in

Gene Mutation Mutation taster DDIG-in

chr6, 158571484C > CCATG g.17828- Disease-causing Disease SERAC1 17829insCATG (prob>0.99) (prob=86.51)

Figure 2. Chromatograms of the affected child’s and his parents’ genome by focusing on the targeted region, including the mutation; A: Father; B: Mother; and C. Affected child

552 Zamani, M., et al. (2020). rs797045105 is the MEGDEL-causing Variant. BCN, 11(4), 549-556. Basic and Clinical July, August 2020, Volume 11, Number 4

Figure 3. ariation location in cDNA. A. Chromatogeram of cDNA sequence. B. Schematic presentation of insertion mutation in DNA & spliced mRNA/cDNA

3.2. Variant characteristics SERAC1 protein (NP_116250) has 654 amino acids, including 32-54 amino acids in transmembrane regions, Following WES data analysis, we identified g.17828- 386-592 as pimeloyl-ACP methyl ester carboxylesteras- 17829insCATG homozygous single-nucleotide varia- es, and 396-540 as /esterases. Up to now, most of tion in the patient in the SERAC1 gene. the reported mutations occurred in the lipases/esterases.

There are 25 reported variants in the ClinVar data- 3.3. Sanger sequencing analysis of genomic am- base (https://www.ncbi.nlm.nih.gov/clinvar/) for the plification SERAC1 gene, including 8 benign, 3 likely benign, 3 likely pathogenic, and 11 pathogenic variants (Table 3). We proved the result of WES of the patient using Sanger The present variant, rs797045105 (Table 2, g.17828- sequencing. Also, allele segregation was checked by par- 17829insCATG) reported in ClinVar as likely pathogen- ents’ genome amplification and sequence assessment of ic variant provided by clinical testing. the targeted region included the variation. The results showed that the patient was homozygous and his parents were heterozygous for the present variant (Figure 2).

Table 3. SERAC1 variants in ClinVar database

Variant Condition (s) Clinical Significance 2-BP DUP, 1627TC MEGDEL syndrome Pathogenic 3-BP DEL, 1435CTT MEGDEL syndrome Pathogenic 4-BP DEL, 1167TCAG MEGDEL syndrome Pathogenic IVS13DS, G-C, +1 MEGDEL syndrome Pathogenic NM_032861.3:c.1916G>C (p.Arg639Pro) Not provided Pathogenic NM_032861.3c.1628_1629dupCT (p.Val544Leufs) Not provided Pathogenic NM_032861.3c.1501+3_1501+6delAAGT Not provided Likely pathogenic NM_032861.3:c.1347_1350dupATCT (p.Val451Ilefs) Not provided Pathogenic NM_032861.3:c.1126C>T (p.Gln376Ter) Not provided Pathogenic NM_032861.3:c.442C>T (p.Arg148Ter) MEGDEL syndrome Pathogenic NM_032861.3:c.265_265+1insCATG MEGDEL syndrome Likely pathogenic NM_032861.3:c.202C>T (p.Arg68Ter) MEGDEL syndrome Pathogenic NM_032861.3:c.21C>G (p.Cys7Trp) Not provided Likely pathogenic

Zamani, M., et al. (2020). rs797045105 is the MEGDEL-causing Variant. BCN, 11(4), 549-556. 553 Basic and Clinical July, August 2020, Volume 11, Number 4

3.4. cDNA analysis gous in the parents. Also, the identified variant was not present in 700 exome files sequenced from 700 referred We demonstrated the insertion of nucleotides in the patients to our laboratory in the southwest of Iran. transcripts of SERAC1 using real-time (RT)-PCR and Sanger sequencing of cDNA product of SERAC1 tran- In conclusion, rs797045105, which has been reported scripts (Figure 3). clinically in the ClinVar database as a likely pathogenic variant could be considered as a pathogenic variant caus- 3.5. ORF finder ing MEGDEL syndrome. The variant was located is in the boundary of exon 4 and introns 4-5. Interestingly, We used the Open Reading Frame (ORF) Finder to Sanger sequencing of SERAC1 cDNA confirmed the search potential protein-encoding segments of the wild insertion of nucleotides in the spliced transcripts of the type and mutant cDNA of SERAC1. The result of ORF gene; therefore, it is predicted to be pathogenic due to Finder showed that the mutant sequence cannot produce the production of p.Gly89fs. SERAC1 encodes a protein correct protein-coding frames. with a serine- domain. This variation disrupts the potential of SREAC1 mRNAs for coding this domain. 4. Conclusion Our results are in line with many studies conducted Neurometabolic disorders are complex because they recently regarding the effectiveness of WES as an ap- are genetically heterogeneous, with different genetic de- propriate tool for the diagnostic of highly heterogeneous fects that give rise to clinically indistinguishable pheno- neurometabolic disorders. types. Finding more pathogenic mutations is effective to identify the disease characteristics and make a progress Ethical Considerations in its diagnosis and treatment. WES is a useful approach in the diagnosis of such complex disorders (Kaname, Compliance with ethical guidelines Yanagi, & Naritomi, 2014; Rabbani, Tekin, & Mahdieh,. 2014, Tarailo-Graovac, et al., 2016). This research study was approved by the Ahvaz Jundis- hapur University of Medical Sciences Institutional Review The 3-MGA-uria type IV, a heterogeneous group of Boards. For WES studies, informed consent was obtained. inborn metabolic errors with primary mitochondrial and All experiments were performed in accordance with rel- endoplasmic reticula impairment, leads to a spectrum of evant guidelines and regulations. multisystem defects. Various enzymes, structural , cellular transport proteins, and other constituents are re- Funding sponsible for neurometabolic disorders including 3-MGA- uria (Wortmann, et al., 2013; Wortmann, et al., 2013). This research did not receive any specific grant from funding agencies in the public, commercial, or not-for- Recently, SERAC1 mutations have been found to cause profit sectors. MEGDEL syndrome (3-MGA-uria type IV). The symp- toms of the disease are central nervous system involve- Authors' contributions ment, neurological deterioration, microcephaly, senso- rineural hearing loss, encephalopathy, dystonia, optic Conceptualization: Mina Zamani, Hamid Galehdari, Ghol- atrophy, and abnormity in the range of urine 3-MGA, amreza Shariati and Alireza Sedghat Mina Zamani; Writ- plasma lactate, alanine, and cholesterol (Wortmann, et ing – original draft: Mina Zamani, Tahereh Seifi, Jawaher al., 2012, Tort, et al., 2013). SERAC1 has a role in the Zeighami, Neda Mazaheri and Hamid Galehdari; Data analy- biosynthetic process of and is an impor- sis: Mina Zamani, Tahereh Seifi, Jawaher Zeighami, Emad tant player in the mitochondria and endoplasmic reticu- Jahangirnezhad; Data collection: Minoo Gholamzadeh; ac- lum functions. quired the data.

In the present study, we carried out WES to investi- Conflict of interest gate the causative genetic mutations in an Iranian pa- There is no conflict of interest regarding the publication tient suspicious of MEGDEL syndrome. Following of this article. data analysis, we identified a frameshift homozygous variation, g.17828-17829insCATG (reported before as rs797045105, in the patient. This variant was heterozy-

554 Zamani, M., et al. (2020). rs797045105 is the MEGDEL-causing Variant. BCN, 11(4), 549-556. Basic and Clinical July, August 2020, Volume 11, Number 4

Acknowledgments

We acknowledge patient and his parents for their coop- eration and participation in the study. We appreciate all the members of Narges Genetic lab.

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